**3. VOCs biomarkers in exhaled breath**

#### **3.1 Overview of breath analysis**

Breath analysis can be considered as a potentially tool for the diagnosis and study of medical diseases. Respiratory analysis has been used in medicine since ancient times, from the time of Hippocrates (460–370 BC), when ancient Greek doctors assessing human health using human breath aroma [34–39]. The first breath analysis was conducted by Lavoisier in 1782, this showing that carbon dioxide from breathing is a product of combustion produced in the body [2, 38–40]. Respiratory studies continued later, when it was shown that human respiration contains VOCs, important in assessing human health [3–5]. The first invention that use gaseous compounds exhaled breath to assess human health was made in 1931 by Dr. Rolla N. Harger. He invented the 'drunkometer a breath-alcohol test, used to determine the alcohol concentration and has been widely used since 1938. An important step in breath analysis was conducted by Pauling et al. in 1971 when using a gas chromatograph were able to detect more than 200 VOCs in human breath [40]. Many studies on exhaled breath have been carried aiming to characterize these VOCs, and the studies carried out of Phillips estimated 1259 compounds in normal subjects in 1997 [41], and over 3000 compounds in 1999 [42]. The analysis of exhaled air is under investigation as a promising tool for express and noninvasive analysis of biochemical processes in the human body. The techniques currently used are promising to translate into clinics not for a specific disease diagnostic but more for providing information about biochemical processes that arise from underlying diseases. Nowadays, there are several commercially available devices for monitoring of breath biomarkers, such as 13C used to diagnose Helicobacter pylori [43], CH4 in breath accompanied by electrochemical sensors to detect H2 and O2 for diagnosis of gastrointestinal disorders [44], asthma detection by exhaled nitric oxide, breath alcohol testing [45], lung cancer detection [46], fructose malabsorption with hydrogen breath test, monitoring uptake of disinfection by-products following swimming [47] chronic kidney disease (CKD) and diabetes mellitus [48].

#### **3.2 Exhaled breath**

Human respiration is a gaseous mixture, and the main compounds are nitrogen, oxygen, carbon dioxide, inert gases, water vapor. In addition to these compounds that are found in high concentration in respiration, VOCs and inorganic molecules can also be found [49]. The presence of the latter in the composition of respiration depends on several external or internal factors. Thus, the composition of human

respiration is influenced both by exogenous compounds from exposure of the human body to various external environmental factors, and from endogenous compounds resulting from biological processes produced in the human body (in the lung tract, blood or peripheral tissues) [50].

Humans breath is a mixture of nitrogen, oxygen, carbon dioxide, inert gases, water vapor and thousands of VOCs traces and inorganic molecules [49]. The composition of breath contains exogenous compounds that originate from environmental exposures, and endogenous compounds that are produced by biological processes in the human body (in the pulmonary tract, blood, or peripheral tissues) [50]. The complex matrix of breath varies from each person both quantitatively and qualitatively. The development of different methods for gas samples analysis has allowed the detection of gaseous compounds traces from respiration, and research has shown that the most common VOCs in human respiration are nitric oxide (10–50 ppb), 1–20 ppb), ammonia (0, 5–2 ppm), carbon monoxide (0–6 ppm), hydrogen sulfide (0–1.3 ppm), acetone (0.3–1 ppm), methane (2–10 ppm), pentane (0–10 ppb) etc. [51–56].

#### **3.3 Breath biomarkers**

Biomarkers are chemicals, usually VOCs, which indicate the normal or abnormal process that take place in the human body and can suggest the presence of a disease or a recent exposure to a drug or an environmental pollutant. The most important breath biomarkers are ammonia, acetone, isoprene, nitic oxide, hydrogen sulphide, methane, ethane and pentane [30–42, 51–58].

#### *3.3.1 Ammonia*

Ammonia (NH3) is very important for the human body and is involved in many physiological processes. The ammonia originates from the catabolism of the amino acids (which are produced mainly by degradation of proteins), can penetrate the blood-lungs and appears in the exhaled breath. Ammonia is absorbed into the portal circulation, taken up by the liver and converted to urea by the urea cycle. Urea cycle is the metabolic pathway that converts urea nitrogen excretion from the body.

Ammonia (NH3) is highly toxic to humans, it is converted into urea which is non-toxic, highly soluble and easily excreted by the kidney. Urea is formed in the urea cycle from NH4 + , CO2, and the nitrogen of aspartate [59, 60]. The cycle occurs mainly in the liver. Ammonia travels to the liver from other tissues, mainly in the form of alanine and glutamine and is released from amino acids in the liver by a series of transamination and deamination reactions. Urea cycle enzyme deficiency will result in insufficient elimination of NH4 + or hyperammonemia which leads to central nervous system deterioration in the form of mental retardation, seizure, coma, and death [60]. The deamination of amino acids, transamination of most amino acids with α-ketoglutaric acid to form glutamic acids, and operation of glutaminase enzyme in the kidney represent the main source of ammonia in the human body. In addition to these sources of ammonia, this can be produced during purine and pyrimidine catabolism, by the action of intestinal bacteria on the non-absorbed dietary amino acids, or by the action of monoamine oxidase enzyme. Therefore, ammonia can be considered to be an important biomarker monitored in the blood, urine, saliva or breath [57–65]. The ammonia concentration is dependent on a range of factors including the health status of the patient, the route of sampling (nasal or oral), contribution from oral bacteria, diet, pharmaceutical use, physical activity and levels of metabolic activity. High levels of ammonia are associated with a variety of pathological conditions, such as hepatic and renal dysfunction, Reye's syndrome, errors

**153**

**detection**

*Organic Volatile Compounds Used in Type 2 Diabetes DOI: http://dx.doi.org/10.5772/intechopen.94752*

*3.3.2 Ethylene*

molecules [71, 72].

radicals, HO·

(ROS), such as superoxide radicals, O2

in the metabolism of urea cycle (urea cycle disorders, UCD) and is also a potential biomarker in exercise physiology and studies of drug metabolism. Normal concentration of ammonia in exhaled breath is 50–2000 ppb (parts per billion) [61–66].

Ethylene (C2H4) is produced by the oxidation of cellular lipids [67, 68]. The relation among ethylene, free radicals, and diabetes can be explained by the oxidative stress. The free radicals attack cellular biomembranes causing cell damage and even cell death [69, 70]. A free radical is an unstable and highly reactive molecular species with an unpaired electron that can donate or accept an electron from other

There are many types of radicals, but the species highly unstable and concern in biological systems are derived from oxygen and known as reactive oxygen species

cant cell damage, damage known as oxidative stress. The term "oxidative stress" (OS) is defined as the imbalance between the production of free radicals and the body's ability to defend itself [18, 19]. This process is kept in balance by the antioxidant defense system. Certain chronic diseases such as atherosclerosis, cancer, diabetes, rheumatoid arthritis, post-ischemic perfusion injury, myocardial infarction, cardiovascular disease, chronic inflammation, stroke and septic shock, aging and other degenerative diseases in humans can be caused or their process it can be accelerated by the appearance of oxidative lesions at the biomolecular level in (lipids, proteins or DNA) when there is an imbalance in the production of free radicals [18, 19, 69–72]. The human body contains a high percentage of lipids (including polyunsaturated fatty acids - PUFA) vulnerable to free radical attack. Lipid peroxidation (LP) occurs as a result of oxidative degradation of polyunsaturated fatty acids induced by free radicals. Therefore, in LP process a peroxidative sequence is initiated by the attack of any free radical species which can extract a hydrogen atom from the group of methylene (CH2), together with an electron on the carbon atom (•CH). By molecular rearrangement the resulting carbon radical is stabilized and is produce a conjugated diene that formed a lipid peroxyl radical (LOO•). But, the propagation of LP continues because these radicals can still extract hydrogen atoms from other lipid molecules to form the lipid hydroperoxides (LOOH). The LP process is finished with the end products that includes malondialdehyde, 4-hidroxinonenal and hydrocarbons, such as pentane, ethane and ethylene [18, 19, 68–74]. The stable end-products of LP, such as ethane, ethylene and pentane are suitable for the estimation of cell damage, because these species are excreted in the breath within a few minutes of their formation in tissues. Excess ethylene in the exhaled breath is associated with biochemical events around LP and can be considered a direct measurement of oxidative stress [67–69, 74]. Ethylene was one of the first breath compounds studied, being reported to range between 3 and 100 ppb [75, 76].

[70–72]. Under stress, ROS levels increase and this can lead to signifi-


·

**4. Laser photoacoustic spectroscopy as a method for breath VOCs** 

The analysis of trace gases from human breath for medical monitoring and diagnostics and require gas sensors characterized by high sensitivity and selectivity (to avoid interference from other potential interfering species), multi-component capability, real time measurements, large dynamic range, in situ measurements, ease to use. Laser photoacoustic spectroscopy (LPAS) is sufficiently sensitive and

in the metabolism of urea cycle (urea cycle disorders, UCD) and is also a potential biomarker in exercise physiology and studies of drug metabolism. Normal concentration of ammonia in exhaled breath is 50–2000 ppb (parts per billion) [61–66].
